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. 2013 Feb 21;49(4):704-18.
doi: 10.1016/j.molcel.2012.12.016. Epub 2013 Jan 24.

A core chromatin remodeling factor instructs global chromatin signaling through multivalent reading of nucleosome codes

Sujit S Nair  1 , Da-Qiang Li, Rakesh Kumar
Affiliations

A core chromatin remodeling factor instructs global chromatin signaling through multivalent reading of nucleosome codes

Sujit S Nair et al. Mol Cell. .

Abstract

ATP-dependent NuRD repressor complexes involve combinatorial assembly of its subunits. However, the mechanism of gene transcription by MTA1/NuRD remains enigmatic. Here we report that MTA1 methylation by G9a methytransferase and demethylation by LSD1 determines the nucleosome remodeling and transcriptional outcome. Contrary to the current static repressor model of the NuRD complex, we discovered that MTA1 association with nucleosomes and corepressor/coactivator complexes is dynamic. While methylated MTA1 is required for the NuRD repressor complex, demethylated MTA1 recognizes the bivalent histone H3K4-AcK9 mark and recruits coactivator NURF-trithorax remodeling complex in a signaling-dependent manner. MTA1's lysine 532 methylation represents a molecular switch as methylated and demethylated MTA1 nucleate NuRD or NURF complexes with opposite functions in a cyclical manner. In addition, MTA1 possesses an inherent histone amplifier activity with an instructive role in impacting the epigenetic landscape, providing a new perspective to the molecular governance of dual coregulator functions of a master coregulator.

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Figures

Figure 1
Figure 1. MTA1 is a methylated component of the NuRD complex
(A) Salt solubilization assay using nuclei from untreated, MAT1 or TSA treated the HeLa cells. Salt soluble and resistant fractions were analyzed using anti-MTA1 and MTA2 antibodies (see also Figure S1A for analysis using anti-CHD3, -CHD4, -MBD and -Rbp antibodies). See also Figure S1A (B) HeLa cells were labeled with 50uCi of (L-Methyl-3H) methionine in presence of protein synthesis inhibitor and incubated for 5h at 37°C. Cell lysates (~1mg) was used for immunoprecipitation using IgG or anti-MTA1 antibody. Immunoprecipitated proteins were electrophoresed in 10% SDS-PAGE, transferred to nitrocellulose membrane and developed by fluorography. Western blot for Anti-MTA1 serves as loading control. See also Figure S1B (C) Sequence alignment of MTA1 highlighting a conserved G9a methylation site in bold. (D) In vitro methylation assay using GST-G9a and His-MTA1, -K532R, and –K631R and bulk histones, S-Adenosyl-L-(methyl-3H)-methionine. Reaction products were analyzed by SDSPAGE and fluorography. See also Figure S1C. (E) Untreated (control), Bix-treated (0.5–1.5µM), control-siRNA and G9a- siRNA treated HeLa cells were labeled with L-Methyl-3H) methionine in presence of protein synthesis inhibitor and incubated for 5 hr at 37°C. Cell lysates (~1mg) was used for immunoprecipitation using IgG or anti-MTA1 antibody. Immunoprecipitated proteins were electrophoresed onto SDS-PAGE, transferred to nitrocellulose membrane and developed by fluorography (top panel) to confirm MTA1 methylation status. Western blot for MTA1 serves as loading control (Second panel from top). Lines, different gels from the same experiment. (F) In vitro methylation using indicated peptide substrates and recombinant G9a. Reactions contained 50 mM glycine (pH 9.6), 2.0 µM [methyl- 3H] AdoMet, 0.50 µM G9a, and 60 µM peptide and were incubated at 37°C for 45 min. Reaction mixtures were precipitated using TCA and quantitated by liquid scintillation. Values are represented as tritium label (CPM)/µg of peptide. Error bars represent SD. (G) In-vitro methylation assay using Flag-G9a enzyme immunoprecipitates, bulk histones and increasing concentrations of His-MTA1 (1×= 0.5ug). Reaction products were analyzed by fluorography and immunoblotting analysis for anti-H3Me2K9 and histone H3. 1/10th of reaction mixture was electrophoresed on a separate gel to confirm the presence of G9a and MTA1 using anti-Flag and anti-His antibodies. (H) Recombinant G9a and GST-MTA1 domains were incubated in HMTase buffer containing 3H-labeled AdoMet and 5µg of histones. Reaction was carried out for 1 hr at 37°C and stopped using SDS-loading buffer and analyzed by fluorography. G9a inhibitor, Bix-01294 (1µM).
Figure 2
Figure 2. Methylated MTA1 directs functional association of the NuRD components
(A) HeLa cells were transfected with T7-MTA1 or methylation-deficient T7-K532R and Flag- G9a. T-7 immunoprecipitates from the nuclear lysates were analyzed by immunoblotting using indicated antibodies. Methylation status of the precipitated MTA1 was determined using anti-methyl lysine antibody (Me-K) in an identical gel. WCL,lysates from transfected cells. (B) Nuclear lysates from the HeLa cells transfected with T7-MTA1 and Flag-G9a were immunoprecipitated using T7-affinity resin. Immunoprecipitate material was analyzed by immunoblotting using indicated antibodies. WCL, lysates from untransfected HeLa cells. Lane 4, CHD4-immunodepleted nuclear lysates. (C) Streptavidin-biotin peptide pull down assay was performed using biotin-tagged control, unmodified, or methylated MTA1 peptides (with mono and dimethyl-lysine substitution at K532) and HeLa cell nuclear extracts. Peptide bound proteins were eluted and analyzed on polyacrylamide gels using indicated antibodies. Lines, different gels from the same experiment. (D) Total RNA was isolated from HeLa cells expressing T7-MTA1 or -MTA1-K532R and 1ug of RNA was reverse transcribed and used in a quantitative PCR reaction using specific primers for endogenous p21/WAF1. Data is presented as fold induction over 18 sRNA (internal control). Error bars represent SD. See also Figure S2C. (E&F) Chromatin prepared from the HeLa cells were subjected to ChIP analysis at p21/WAF1 promoter, using anti-MTA1 (E) or ChIP analysis using anti-Methyl-lysine antibody (F) followed by re-ChIP using indicated antibodies.. Recruitment profiles were generated in quantitative-PCR assay specific primers encompassing MTA1 recruitment region on p21-WAF1 promoter. Values are represented as fold recruitment/IgG. **p<0.01,***p<0.001, ***p<0.005. Error bars represent SD. (G) Quantitative ChIP relative to IgG control at p21/WAF1 promoter using indicated antibodies and chromatin from MTA1-WT and KO cells transfected with T7-MTA1 or -methylation deficient K532R. Methylation status of MTA1 at target chromatin is evaluated using anti-Methyl lysine antibody. ***p<0.005. Error bars represent SD. (H) Chromatin prepared from the HeLa cells were subjected to ChIP analysis onto p21/WAF1 using MTA1-MeK532 antibody followed by Re-ChIP using indicated antibodies. Quantitative-PCR using specific primers encompassing MTA1 recruitment region on p21-WAF1 promoter were used to generate recruitment profiles. Values are represented as fold recruitment/IgG. ***p<0.001, ***p<0.005. Error bars represent SD.
Figure 3
Figure 3. MTA1 methylation status directs NuRD dependent chromatin remodeling
(A) In vitro demethylation assay using G9a methylated His-MTA1, Methylated histones and LSD1. COS1 cells were transfected with Flag-LSD1-WT (1×=2.5µg) and -demethylase deficient LSD-661A. Flag immunoprecipitates were used in demethylase reaction. Reaction products were analyzed by fluorography. (B) Ni-NTA-bound His-MTA1 or His-MTA1-K532R was used in methyltransferase assay with G9a. Ni-NTA beads were washed several times in the immunoprecipitation buffer, bound nonmethylated His-MTA1; methylated His-MTA1 and His-MTA1-K532R were incubated with 1 mg of the HeLa cells’ nuclear extracts. Bound proteins were eluted in the SDS-loading buffer and analyzed onto SDS-PAGE using indicated antibodies. An aliquot of reaction was electrophoresed on separate gel and analyzed using anti- methyl lysine antibody to determine MTA1 methylation status. (C) HeLa cells expressing T7-MTA1 or -MTA1-K532R were treated with either control or G9a siRNA or transfected with Flag-G9a and -LSD1. Nuclear lysates were incubated with anti-T7- antibody or control IgG and analyzed by immunoblotting using indicated antibodies. Methylation status of MTA1 under all conditions was evaluated by fluorography (top panel). Western blot for Anti-MTA1 serves as loading control (Second panel from top). Lines, different gels from the same experiment. (D) HeLa cells expressing various expression vectors were treated with TSA and sodiumbutyrate for 12 hr. Nuclear lysates were immunoprecipitated by T7 affinity resin, resolved onto a SDS-PAGE and immunoblotted with the indicated antibodies. Lines, different gels from the same experiment. (E) ATP binding of T7-MTA1, -MTA1-K532R, and -MTA1-K631R precipitated from HeLa cells with 1µCi of γ-32ATP. ATP binding and hydrolysis was evaluated using formaldehyde-urea gels. See also Figure 3C. (F) ATP binding of the endogenous MTA1 immunoprecipitated from the HeLa cells. IgG or MTA1 immunoprecipitates from CHD4-immunodepleted or Bix-treated nuclear lysates were incubated with either 1 µCi of γ-32ATP alone or in combination with 3-fold excess cold non-hydrolysable ATP. ATP binding and hydrolysis was evaluated using formaldehyde-urea gels. Presence of CHD4 in MTA1 immunoprecipitates were analyzed using anti-CHD4 antibody. (also see Figure S3C for immunoblotting using anti-T7, -G9a, and -LSD1 antibodies). Lines, different gels from the same experiment. (G) In vitro mono-nucleosome mobilization and chromatin remodeling assay using reconstituted nucleosomes and T7 immunoprecipitates containing methylated and de-methylated MTA1 containing complexes from the COS-1 cells transfected with indicated expression plasmids.
Figure 4
Figure 4. MTA1 is a reader of modified histones
(A-C) Peptide pull down assays were carried out using the HeLa cells’ nuclear extracts and biotinylated histone H3, H2B and H4 peptides (aa1–22) (A)or biotinylated Histone H3 (aa1–11, aa12–21, aa22–44) peptides (B) or N-terminal histone H3 peptides with specific methyl and acetyl modifications (C). HP1-alpha pulldown, was positive control for H3K9Me3 binding. The material was immunoprecipitated using Streptavidin where indicated. See also Figure S4. (D) Histone binding assay using T7-WT MTA1 or T7-MTA1-K532R from the COS1 cells and core histones from the HeLa cells. Bound histones were electrophoresed onto a 15% PAGE and analyzed using indicated antibodies. Immunoblotting analyses using anti-flag (G9a and LSD1) and anti-T7 is shown here. The material was immunoprecipitated using an anti-T7 antibody where indicated. (E) In vitro translated 35S labeled MTA1 domains were used in a peptide pull-down assay using various histone H3 biotinylated peptides. (F) Biotinylated peptide pull down assay was performed using control unmodified and biotinylated Histone H3Acetyl lysine-9 and Histone H3 Methyl lysine-4-Acetyl-lysine-9 peptide and HeLa cell nuclear extracts. (G) In vitro methylation assay with G9a enzyme and GST and GST-MTA1-441–715. Methylation of GST-MTA1-441–715 was confirmed by fluorography (left panel). Inclusion of Bix (1µM) completely abolished methylation. Equal amount of GST, unmethylated and G9a methylated MTA1-441–715 was used in a streptavidin-biotin peptide pull-down assay. 50ng of biotinylated histone H3K4AcK9 was incubated with equal amount of GST, unmethylated and G9a-methylated GST-MTA1-445–715 in histone binding buffer (20mM Hepes, ph 8.0, 200mM KCL, 0.5% TritonX-100, 0.2mM EDTA) for 3 hr at room temperature. Beads were washed with high stringent wash buffer (histone binding buffer supplemented with 400 mM KCL) and bound proteins were eluted using glycine buffer (pH:3.0). Eluted proteins were analyzed onto a 10% SDS-PAGE and transferred to nitrocellulose membrane and stained with Ponceau. Blots were scanned and further treated with fluorgraphy reagent to detect methylated GST-fusion proteins.
Figure 5
Figure 5. MTA1 demethylation recruits co-activator functions
(A) Total RNA from HeLa cells transiently expressing T7-MTA1 or -MTA1-K532R were transfected was reverse transcribed and used in a quantitative PCR reaction using specific primers for endogenous Pax5. Data is represented as fold induction over 18 sRNA (internal control). Where indicated small molecular inhibitor of G9a, Bix-01294 and pargyline-hydrochloride a specific small molecule inhibitor of LSD1 were used at a final concentrations of 1µM and 1mM, respectively. Error bars represent SD. See also Figure S5A (B) Flag-LSD1 or –LSD1-K661A from the COS1 cells were used in the histone demethylase assay. Where indicated recombinant His-MTA1 was included in the assay. Histones from demethylase reaction were analyzed independently using anti-H3Me2K4 and anti-H3-Me2K9 antibodies. Lines, different gels from the same experiment. (C) HeLa cells were transfected with the indicated expression vectors were used for ChIP assay to evaluate changes at the Pax5 promoter, using anti-T7 antibody followed by Re-ChIP using indicated antibodies. Recruitment profiles were generated using quantitative-PCR and specific primers encompassing MTA1 recruitment region on Pax5 promoter. Values are represented as fold recruitment/IgG. **p<0.01.Error bars represent SD. See also Figure S5B. (D) Quantitative ChIP relative to IgG control at the Pax5 promoter using indicated antibodies and chromatin from MTA1-WT and MTA1-KO cells transfected with T7-MTA1 or -MTA1- K532R. (also see Supplementary Figure 4C). ***p<0.005. Error bars represent SD. See also Figure S5C. (E) Histone acetyltransferase assay (HAT) using immunoprecipitates from Ni-NTA or streptavidin-biotin pull down from the HeLa cells nuclear extracts were incubated with His-MTA1 protein or biotinylated peptides. Lines, different gels from the same experiment.
Figure 6
Figure 6. MTA1 associates with the components of the NuRD and NURF complexes in a cyclical and signaling dependent manner
(A) MCF-7 cells expressing T7-MTA1 or –MTA1-K532 constructs were serum starved for 48 hours followed by serum stimulation for indicated time points, and lysates were immunoprecipitated with T7-affinity-resin. Bound immunoprecipitates (retained proteins) were analyzed separately on a polyacrylamide gel followed Western blot analysis using indicated antibodies. Methylated MTA1 binds co-repressor complex while demethylated MTA1 recruits activator complex. Lower panel from a different gel from the same experiment. (B) Chromatin was prepared from the MCF-7 transfected with the T7-MTA1, MTA1-K532R. Quantitative ChIP with T7 antibody followed by re-ChIP with indicated antibodies is represented relative to IgG control at EZH2, CHD4, IL-12 and CHD8 promoters. Error bars represent SD. (C) Chromatin was prepared from MCF-7 transfected with MTA1-WT, MTA1-K532R and serum treated at indicated time points. Quantitative ChIP with T7 antibody followed by Re-ChIP with indicated antibodies is represented relative to IgG control at EZH2 promoter. Values are represented as fold recruitment/IgG. Green colored bars in graph represent status of methylated MTA1 at indicated time points . Error bars represent SD. See also Figure S6A (D) Total RNA from MCF-7 cells transfected with the indicated expression vectors were used reverse transcribed and subjected to semi-quantitative PCR using specific primers to detect EZH2 and CHD8 mRNA levels.
Figure 7
Figure 7. MTA1 engages with the NuRD and NURF complexes in a cyclic manner
(A) Cells were stimulated with serum for indicated time points. Recruitment profiles of MTA1 and repressor/activator marks at EZH2 promoter was generated by ChIP using a MTA1-antibody followed by Re-ChIP using MTA1 methylation site specific (MTA1-MeK532) antibody. Values are represented as fold recruitment/IgG. Error bars represent SD. See also Figure S7. (B) Model of MTA1 dual-coregulator action (Also graphical abstract). At repressive chromatin target, G9a methylated MTA1 is associated with histone H3 aa11–21. Methylation of MTA1 serves as a docking site for CHD4. Binding of CHD4 directs a combinatorial association of NuRD chromatin remodeling complex. MTA1 potentiates G9a methyltransferase activity on HDAC2 deacetylated nucleosomes to create a repressive H3K9Me2 environment. During gene activation cycle, LSD1 is recruited to the NuRD repressor complex and demethylates MTA1. Loss of MTA1-methylation destabilizes the NuRD repressor complex. Increased affinity of demethylated MTA1 for H3K9Me2 triggers rapid demethylation of H3-K9Me2 mark by LSD1 and acetylation by p300/CBP. MTA1 functions as an epigenetic reader of H3K4AcK9 and engages in a co-activator function. Methylated and demethylated states of MTA1 engage distinct histone modifications and factors to facilitate gene repression (NuRD) or activation (NuRF) events.
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